The present invention relates to the fields of molecular biology and signal transduction. More specifically, the invention provides nucleic acid molecules, proteins and antibodies useful as targets for screening therapeutic agents that regulate-cell cycle progression and apoptotic cell death.
Several publications are referenced in this application by numerals in parenthesis in order to more fully describe the state of the art to which this invention pertains. Full citations for these references are found at the end of the specification. The disclosure of each of these publications is incorporated by reference herein.
Programmed cell death (PCD) is a genetically controlled active process which removes cells that are harmful or no longer necessary for the organism. Morphologically, cells undergoing PCD suffer cytoplasmic membrane blebbing, chromatin condensation and disassembly into membrane-enclosed vesicles, called apoptotic bodies, and DNA fragmentation. This constellation of morphological features has been termed apoptosis. Although the terms PCD and apoptosis were originally coined to define different concepts, today they are used almost interchangeably. For the purpose of this application, the term apoptosis will be used exclusively.
Apoptosis plays an important role in development as well as in tissue homeostasis in adult animals. Tissues of adult animals undergo continuous renewal characterized by cellular proliferation, differentiation and death by apoptosis. Defects that increase the rate of cell death lead to degenerative diseases while defects that decrease it lead to cancer. A classical example of the latter are follicular B cell lymphomas that carry a t14;18 translocation and overexpress the Bcl-2 protooncogene.
As eucaryotic cells grow and divide, they progress through an ordered cycle of events in which chromosome replication (S-phase) and mitosis (M-phase) are separated by two gap phases (G1xe2x86x92Sxe2x86x92G2xe2x86x92Mxe2x86x92G1). The gap phases are punctuated by checkpoints, at which progression stalls if certain requirements are not met. The G1 checkpoint prevents S-phase if the cell has sustained damage to its DNA, and the G2 checkpoint ensures complete DNA-replication and chromosome integrity prior to mitosis. The timing of these events is coordinated by the sequential activation and inactivation of a series of kinase complexes consisting of a catalytic subunit (the cyclin dependent kinase or cdk) and a regulatory subunit (the cyclin). Each cell cycle phase is characterized by the activity of a specific set of cyclin/cdk complexes. In mammalian cells, cyclins D/cdk4 (or cdk6) and cyclin E/cdk2 are required for progression through G1 and entry into S phase, cyclin A/cdk2 is required during S phase and the mitotic cyclin B/cdk1 is required during G2 and mitosis. Waves of phase-specific CDK activities are generated by at least three means: undulations in the availability of catalytic subunits; regulatory phosphorylation and dephosphorylation of the catalytic subunits; and modulation of the abundance of inhibitory molecules.
The regulation of the cell cycle in cycling cells depends on oscillations of the activity of these and perhaps other molecules, controlled by an internal clock. In addition however, the mammalian cell cycle responds to external mitogenic signals. The response to such signals is restricted to the G1 phase of the cycle before the cells can commit to replicate their chromosomal DNA. The loss of mitogen dependency late in G1 marks the restriction point (G1 checkpoint or START). Signals required for, or modulating G1 progression are transduced via several signalling pathways.
Apoptosis and the cell cycle are intricately interconnected processes. Thus, dividing cells that do not fulfill the requirements to traverse the G1 or G2 checkpoints undergo either cell cycle arrest or apoptosis. As a result, death by apoptosis may occur at highly predictable points in the cell cycle and in response to signals generated by the machinery that monitors the requirements for cell cycle progression. This is exemplified by the outcome of conflicting signals that both stimulate and inhibit progression through the cell cycle. Such signals predictably induce apoptosis. Examples include: 1) the induction of apoptosis by serum starvation in cultured fibroblasts overexpressing c-myc; and 2) the induction of apoptosis by -irradiation or induction of p53 in cells expressing active E2F-1. In addition to these links between apoptosis and the cell cycle, it has also been shown recently that proteins that primarily regulate apoptosis, such as anti-apoptotic or proapoptotic members of the Bcl-2 family, also affect cell cycle progression. Finally, the same signalling molecules may target both regulators of the cell cycle and regulators of apoptosis.
Apoptosis and the cell cycle are fundamental biological processes. Exploring the regulation of these processes has already provided significant insights into the nature and the origin of genetic defects, degenerative diseases and cancer. The present invention provides composition and methods for identifying regulators of apoptotic cell death for the subsequent generation of beneficial, therapeutic agents.
This invention provides novel biological molecules useful for identification, detection and/or regulation of complex signalling events that regulate cell cycle progression and apoptotic cell death. According to one aspect of the invention, a nucleic acid molecule is provided that encodes a Tvl-1 protein between about 250 and 280 amino acids in length, said protein comprising a plurality of ankyrin repeat domains and promoting TNF-xcex1 induced apoptosis. In a preferred embodiment the protein has the amino acid sequence of SEQ ID NO:2. An exemplary nucleic acid of the invention comprises SEQ ID NO:1.
According to another aspect of the present invention, antibodies immunologically specific for the Tvl-1 protein are provided.
Various terms relating to the biological molecules of the present invention are used hereinabove and also throughout the specifications and claims. The terms xe2x80x9cspecifically hybridizing,xe2x80x9d xe2x80x9cpercent similarityxe2x80x9d and xe2x80x9cpercent identity (identical)xe2x80x9d are defined in detail in the description set forth below.
With reference to nucleic acids of the invention, the term xe2x80x9cisolated nucleic acidxe2x80x9d is sometimes used. This term, when applied to DNA, refers to a DNA molecule that is separated from sequences with which it is immediately contiguous (in the 5xe2x80x2 and 3xe2x80x2 directions) in the naturally occurring genome of the organism from which it originates. For example, the xe2x80x9cisolated nucleic acidxe2x80x9d may comprise a DNA or cDNA molecule inserted into a vector, such as a plasmid or virus vector, or integrated into the genomic DNA of a prokaryote or eukaryote.
With respect to RNA molecules of the invention, the term xe2x80x9cisolated nucleic acidxe2x80x9d primarily refers to an RNA molecule encoded by an isolated DNA molecule as defined above. Alternatively, the term may refer to an RNA molecule that has been sufficiently separated from RNA molecules with which it would be associated in its natural state (i.e., in cells or tissues), such that it exists in a xe2x80x9csubstantially purexe2x80x9d form (the term xe2x80x9csubstantially purexe2x80x9d is defined below).
With respect to protein, the term xe2x80x9cisolated proteinxe2x80x9d or xe2x80x9cisolated and purified proteinxe2x80x9d is sometimes used herein. This term refers primarily to a protein produced by expression of an isolated nucleic acid molecule of the invention. Alternatively, this term may refer to a protein which has been sufficiently separated from other proteins with which it would naturally be associated, so as to exist in xe2x80x9csubstantially purexe2x80x9d form.
The term xe2x80x9csubstantially purexe2x80x9d refers to a preparation comprising at least 50-60% by weight the compound of interest (e.g., nucleic acid, oligonucleotide, protein, etc.). More preferably, the preparation comprises at least 75% by weight, and most preferably 90-99% by weight, the compound of interest. Purity is measured by methods appropriate for the compound of interest (e.g. chromatographic methods, agarose or polyacrylamide gel electrophoresis, HPLC analysis, and the like).
With respect to antibodies of the invention, the term xe2x80x9cimmunologically specificxe2x80x9d refers to antibodies that bind to one or more epitopes of a protein of interest (e.g., Tvl-1), but which do not substantially recognize and bind other molecules in a sample containing a mixed population of antigenic biological molecules.
With respect to oligonucleotides, the term xe2x80x9cspecifically hybridizingxe2x80x9d refers to the association between two single-stranded nucleotide molecules of sufficiently complementary sequence to permit such hybridization under pre-determined conditions generally used in the art (sometimes termed xe2x80x9csubstantially complementaryxe2x80x9d). In particular, the term refers to hybridization of an oligonucleotide with a substantially complementary sequence contained within a single-stranded DNA or RNA molecule of the invention, to the substantial exclusion of hybridization of the oligonucleotide with single-stranded nucleic acids of non-complementary sequence.
The nucleic acids and proteins of the present invention are the products of a single gene (Tvl-1). The proteins encoded by the Tvl-1 gene function as adaptors that contribute to the assembly and/or stability of multi-molecular complexes. Since these proteins are present both in the cytoplasm and in the nucleus we propose that they contribute to the assembly/stability of multi-molecular complexes in both subcellular locations. The information disclosed in this application shows that the proteins encoded by Tvl-1 contribute to the assembly of cytoplastic and nuclear complexes involved in apoptosis and cell cycle regulation. Therefore, the nucleic acids proteins and antibodies of the present invention can be advantageously used as targets for development of novel therapeutic agents which regulate cell cycle progression and apoptotic cell death. The Tvl-1 molecules of the invention can also be used as research tools and will facilitate the elucidation of the mechanistic action of the novel genetic and protein interactions involved in the control of cellular proliferation and apoptosis.